The government may own certain rights in the present invention pursuant to NIH grants CA-41081 and CA-28149.
1. Field of the Invention
The present invention relates to improved methods for screening antibody populations to select those populations having cytotoxic potential as immunotoxins. In particular aspects, the present invention relates to indirect assays for screening populations of antibodies employing a screening reagent which includes a toxin-linked univalent antibody fragment.
2. Description of the Related Art
In recent years, conjugates of cell-reactive antibodies and the A chains of toxins such as ricin, diptheria toxin and the like, have become increasingly important in medical applications. For example, such conjugates, referred to as immunotoxins, have been used to kill normal and malignant target cells both in vitro (1-4) and in vivo (5-9). Moreover, immunotoxins have been used both experimentally and clinically in the management or treatment of a variety of diseases or disorders, such as the treatment of autoimmune diseases, various malignancies, and even to purge bone marrow of T-cells or tumor cells before transplantation (reviewed in Ref's 10 and 11).
A significant problem, however, in the development of immunotoxins is the finding that not all monoclonal antibodies make highly toxic immunotoxins. Different monoclonal antibodies used as immunotoxins vary over 10,000-fold in their potency as specific cytotoxic reagents (10,11). It appears that some of the factors which determine the potency of an immunotoxin include the cell surface antigen to which the antibody is directed and its resultant pathway of internalization after binding to the immunotoxin (12-14), the density of the cell surface antigen (15-16), the epitope recognized by the antibody, and the affinity of the antibody utilized (17). Furthermore, different cells expressing the same antigenic determinants may vary in their susceptibility to the same immunotoxin (17-19). Because of this complexity, it is difficult to predict whether any given antibody will make an effective immunotoxin.
Based on the results of studies by Bjorn et al. (18), one can estimate that only one in four monoclonal antibodies, at best, is of sufficient potency (IC.sub.50 =10.sup.-11 -10.sup.-13 M) to be considered for clinical use as an immunotoxin. Previously, in screening monoclonals for their potential use as immunotoxins, it has been necessary to produce and purify large quantities of monoclonal antibodies, couple them to ricin A chain, and purify the resultant immunotoxin. Each immunotoxin must then be tested for its ability to kill an appropriate target cell. This procedure is laborious, expensive and limits the number of monoclonal antibodies that can be evaluated.
Recently, Weltman et al. (20) described a method for potentially screening large numbers of monoclonal antibodies for their efficacy as immunotoxins. This method, called an "indirect immunotoxin" assay, is similar in some respects to indirect immunofluorescence staining. Target cells are treated with primary (1.degree.) unconjugated monoclonal antibodies, washed, and then incubated with a secondary (2.degree.) toxin-linked immunoglobulin antibody having binding immuno-specificity for the primary antibody.
In the Weltman et al. study (20), this method was used to screen 12 monoclonal antibodies specific for a small cell lung cancer line. One monoclonal antibody was reported to induce significant inhibition of protein synthesis in the cell in the indirect assay. However, the validity, sensitivity and specificity of the approaches employed by Weltman et al. was not shown because none of the antibodies were employed directly as immunotoxins. Furthermore, the design of the assay was seriously flawed--because immunoglobulins are immunologically divalent, it is likely that the primary cell-bound monoclonal antibody is being cross-linked. Cross-linking can alter the route of internalization of monoclonal antibodies and thus significantly alter the toxicity of the toxin-linked secondary antibody (21).
Due to the potential lack of specificity and sensitivity in existing indirect assays, there is presently a need for improved assays for screening antibodies. In particular, there is a need to assays which can predict the usefulness of antibodies in the preparation of immunotoxins which do not present the possibility of inaccuracies due to cross-linking of the primary antibody.